In vitro assembly and disassembly of 14-nm filament from Tetrahymena pyriformis. The protein component of 14-nm filament is a 49,000-Dalton protein.
Conditions for in vitro assembly and disassembly of Tetrahymena 14-nm filaments were investigated electron-microscopically by using a crude extract of acetone powder of the cells. The assembly conditions established are: incubation of a protein sample (2 mg/ml) in 5 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 6.6) containing 0.1 mM N alpha-tosyl-L-lysyl-chloromethane hydrochloride (TLCK), 50 mM KCl, 0.6 mM ATP, and 1.2 mM CaCl2 at 30 degrees C for 30 min. The disassembly conditions established are: dialysis of the 14-nm filament suspension (3 mg protein/ml) against Tris-acetate buffer (pH 8.2) containing 5 mM 2-mercaptoethanol, 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), and 0.05 mM TLCK at 4 degrees C for 24 h. The assembly and disassembly were repeatable, and resulted in the exclusive retention of the 49,000-dalton protein. This clearly shows that the previously reported protein component (38,000-dalton protein : FFP-38) of the 14-nm filament is incorrect and the actual component is indeed a 49,000-dalton protein. The present research also showed that the Tetrahymena 14-nm filament bore a strong resemblance to 'intermediate filaments' of mammalian cells with respect to molecular weight, amino-acid composition of the protein component, and size and conditions for assembly and disassembly of the filament.
D000596:Amino Acids / Q000302:isolation & purification; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D003599:Cytoskeleton / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008970:Molecular Weight; D011506:Proteins / Q000302:isolation & purification*; D013696:Temperature; D013769:Tetrahymena pyriformis / Q000378:metabolism* / Q000648:ultrastructure
D000596:Amino Acids; D011506:Proteins